Fig 1.
SEM images and bubble charts of microbes, plants and arthropod-related reads on the adaxial and abaxial leaf surfaces.
The bubble sizes correspond to the relative abundance of reads assigned to bacteria, fungi, plant host and arthropods in the comparative metagenomic analyses. Reduction in bacterial read counts observed on the abaxial surface as compared to the adaxial surface in contrast to Fungi, Viridiplantae and Arthropoda reads on the leaf surfaces of (a) R. excelsa and (b) C. fruticosa. The moon and sun symbols refer to the time of sampling (midnight and noon), while the letters refer to the sampling location in S1 Fig. The SEM images were original images captured by Chee Peng Ng and David Liebl from the IMB-IMCB Joint Electron Microscopy Suite (A*STAR Singapore).
Fig 2.
Dot plots of species richness using Chao-1 index of the adaxial and abaxial leaf surface.
Approximately 95–98% fewer in bacterial species were observed on the abaxial leaf surface of (a) R. excelsa and (b) C. fruticosa in contrast to their adaxial surface. There was also a decrease in fungal species observed on the abaxial surface of (c) R. excelsa and (d) C. fruticosa. All four dot plots showed statistically significant differences between the adaxial and abaxial surface using Mann Whitney test (S2 Table). The photographs were taken by Balakrishnan N.V. Premkrishnan.
Fig 3.
Co-occurrence network analysis of microorganisms on the adaxial and abaxial leaf surfaces.
Of the nine clusters identified, two are highly interconnected (clusters A and B). Microorganisms in both clusters are highly abundant on the adaxial surface but are significantly reduced on the abaxial surface. Cluster A is composed of soil and leaf bacteria while cluster B comprises of wood-rotting fungi, photosynthetic cyanobacteria, thermophilic and radiation-tolerant bacteria. The nodes were selected with a cut-off of Spearman rank correlation coefficient of 0.8 and p-value of 0.01.
Fig 4.
Relative abundance of each microbial co-occurrence cluster.
Comparison of the normalised read counts among the nine microbial clusters (A to I) revealed that the orange cluster, composed of leaf blotch fungi, dominates the leaf surfaces of (a) R. excelsa and (b) C. fruticosa. The leaf blotch fungi were twice as abundant on the abaxial surface compared to the adaxial counterpart. The dense clusters A and B network observed in Fig 3 suggest an interaction among the group of leaf, soil, and radio-tolerant bacteria on the adaxial surface. These results further detail the reduction in microbial diversity on the abaxial surface.
Fig 5.
Reactive oxygen species assay of adaxial and abaxial leaf surfaces.
A more than 2-fold increase of ROS production was observed on the abaxial surface of (a) R. excelsa. However, this phenomenon was absent in (b) C. fruticosa. The fluorescence intensity readings of the ROS assay are listed in S3 Table. The difference in concentration of ROS between adaxial and abaxial surface of R. excelsa was statistically significant using Welch’s t-test (S5 Table). The photographs were taken by Kenny J.X. Lau.
Fig 6.
Surface wettability of leaf surfaces.
The water contact angle was measured from images captured at orthogonal angle. The adaxial and abaxial leaf surfaces of (a) R. excelsa have contact angles of 83° and 95°, respectively. The adaxial and abaxial leaf surfaces of (b) C. fruticosa have contact angles of 102° and 140°, respectively. The abaxial surface is hydrophobic and repels water from its surface. The water droplet images were original images taken by Kenny J.X. Lau using the contact angle meter.